The developmental program that creates segments along the anterior-posterior axis of the Drosophila embryo is a classic system for studying regulation of gene expression, and has provided functional information about conserved regulators that also function in human development and disease. One outstanding question is how important regulatory factors, and networks of factors that act in concert, can evolve new function, since "correct" outputs are essential in every generation for survival. Most of the genes utilized in embryo segmentation in flies have been extensively characterized since genetic tools available in Drosophila make possible profound inquiries into gene function and regulation of expression. Comparative studies using Drosophila and other insects provide additional tools for elucidating the mechanisms underlying evolution of programs that use these factors. The wasp Nasonia has a body plan similar to that of Drosophila, in spite of more than 250 million years of independent evolution, making it a good system for comparative studies. Moreover, Nasonia seems to possess some character of ancestral insects that has been lost in the fly lineage, providing a unique opportunity to gain insight into how gene networks proceed from one state to another. Findings of this nature are valuable for understanding changing gene function in human pathology and disease, where coordinated functional programs deviate pathologically in a way that is sustained. I am using the Nasonia segmentation gene network for comparative studies, specifically the pair- rule class of genes. Although these genes are conserved in function, their regulation by upstream maternal and gap genes place them at a critical point to integrate differences in early developmental strategies between flies and wasps. First, pair rule gene interactions will be analyzed through characterization of expression and functional studies. I will then focus on regulation of the even-skipped promoter in transgenic Nasonia, informed by bioinformatics methods and by extensive studies in Drosophila, to identify specific enhancer elements that coordinate spatial and temporal control of stripe expression in the anterior of the Nasonia embryo. Finally, the molecular controls governing segment identity and growth in the posterior of Nasonia will be sought, to understand ancestral gene network configurations and potentially suggest how subnetworks controlling an embryo's pattern can be unified.